C-Terminal Binding Proteins: Central Players in Development and Disease

BioMol Concepts 2014; 5(6): 489–511

Review

Trisha R. Stankiewicz, Josie J. Gray, Aimee N. Winter and Daniel A. Linseman* C-terminal binding proteins: central players in development and disease

Abstract: C-terminal binding proteins (CtBPs) were initially Introduction identified as binding partners for the E1A-transforming proteins. Although the invertebrate genome encodes one C-terminal binding protein (CtBPs) were originally identi- CtBP protein, two CtBPs (CtBP1 and CtBP2) are encoded fied as binding partners for the adenovirus E1A protein, by the vertebrate genome and perform both unique and suggesting an important function for CtBPs in regulat- duplicative functions. CtBP1 and CtBP2 are closely related ing cell survival (1, 2). CtBPs are evolutionarily conserved and act as transcriptional corepressors when activated by proteins and are present in a diverse number of species nicotinamide adenine dinucleotide binding to their dehy- ranging from terrestrial plants to Caenorhabditis elegans drogenase domains. CtBPs exert transcriptional repres- to humans (3). Whereas the vertebrate genome encodes sion primarily via recruitment of a corepressor complex two highly homologous CtBP proteins, CtBP1 and CtBP2, to DNA that consists of histone deacetylases (HDACs) the invertebrate genome encodes one CtBP transcript. The and histone methyltransferases, although CtBPs can also major splice forms of CtBP1 and CtBP2 function primar- repress transcription through HDAC-independent mecha- ily as transcriptional corepressors. CtBPs associate with nisms. More recent studies have demonstrated a critical various DNA-binding repressors such as basic Krüppel- function for CtBPs in the transcriptional repression of like factor 8 (BKLF8) to recruit chromatin-modifying pro-apoptotic genes such as Bax, Puma, Bik, and Noxa. enzymes to the promoter regions of DNA (4, 5). However, Nonetheless, although recent efforts have characterized some minor CtBP splice variants also mediate various the essential involvement of CtBPs in promoting cellular cytosolic functions (3). survival, the dysregulation of CtBPs in both neurodegen- Genetic knockout studies have demonstrated that erative disease and cancers remains to be fully elucidated. CtBP1 and CtBP2 perform both unique and duplicative functions during development and genetic deletion of Keywords: anoikis; apoptosis; cancer; C-terminal binding CtBPs results in severe developmental defects and embry- proteins; development; neurodegenerative disease. onic lethality (6). Consistent with their role in transcriptional corepression, Grooteclaes and colleagues (4, 7) DOI 10.1515/bmc-2014-0027 identified CtBPs as critical repressors of apoptotic and Received August 11, 2014; accepted October 7, 2014 anoikis gene programs. Subsequent studies have demonstrated that CtBPs bind the viral oncoproteins EBNA3A and EBNA3C, further demonstrating that CtBPs have significant roles in regulation of cellular proliferation and survival (8, 9). Given the critical functions of CtBPs in promoting cell survival, dysregulation of CtBPs has emerged *Corresponding author: Daniel A. Linseman, Research Service, as a potential causative factor underlying neurodegenera- Veterans Affairs Medical Center, Research 151, 1055 Clermont tive diseases as well as cancer. Street, Denver, CO 80220, USA, e-mail: [email protected]; In this review, we summarize current information on Department of Biological Sciences and Eleanor Roosevelt Institute, the structure, function, and regulation of CtBPs. Next, University of Denver, Denver, CO 80208, USA; and Division of Clinical Pharmacology and Toxicology, Department of Medicine and we discuss the involvement of CtBPs in development, Neuroscience Program, University of Colorado Denver, Aurora, CO apoptosis, and anoikis. Finally, we highlight recent data 80045, USA supporting dysregulation of CtBPs in neurodegenera- Trisha R. Stankiewicz and Josie J. Gray: Research Service, Veterans tive diseases and cancer. Given the extensive evidence Affairs Medical Center, Denver, CO 80220, USA; and Department of demonstrating a pro-survival function for CtBPs in both Biological Sciences and Eleanor Roosevelt Institute, University of Denver, Denver, CO 80208, USA non-neuronal and neuronal cell populations, CtBPs are Aimee N. Winter: Department of Biological Sciences and Eleanor emerging as novel molecular targets for the treatment of Roosevelt Institute, University of Denver, Denver, CO 80208, USA diseases characterized by either enhanced apoptosis (i.e., 490 T.R. Stankiewicz et al.: CtBPs in development and disease neurodegeneration) or increased resistance to apoptotic CtBP to execute its corepressor functions (10). CtBPs can cell death (i.e., cancer). also bind to proteins directly via the PXDLS domain to repress their function (15). In addition to the PXDLS-binding pocket, all CtBP family members also contain a surface groove that is Structure and function of CtBPs capable of binding proteins with an RRT-binding motif (16). Like the PXDLS hydrophobic cleft, the RRT-binding Domain structure of CtBPs pocket of CtBPs is predominantly used to bind and recruit members of the corepressor complex, and many CtBP- CtBP1 and CtBP2 are highly conserved and share a sig- interacting proteins (CtIPs) that bind at this site also nificant degree of sequence and structural homology contain PXDLS-binding motifs (10, 16). Thus, each CtBP (Figure 1). Of these two proteins, CtBP1 has been the most monomer contains two binding sites that can be occupied extensively characterized. Although CtBP1 and CtBP2 simultaneously by distinct members of the corepressor possess high sequence homology and structural simi- complex. Together, these two binding sites account for larities, they have been shown to exert both unique and a majority of CtBP interactions with proteins of the core- duplicative effects (6). Despite these diverse functions, pressor complex and repressor proteins that recruit CtBPs however, all CtBP family members share several key char- to specific gene promoters. acteristics that are essential for protein function. Intriguingly, it is now well established that CtBP A hydrophobic cleft known as the PXDLS-binding site proteins share significant sequence homology with 2D is one of the best characterized features of the CtBP family hydroxyacid dehydrogenases, particularly within the RTT- and several CtBP-interacting partners containing PXDLS- binding cleft (Figure 1) (17). Moreover, the dehydrogenase binding motifs have been identified. In many cases, muta- domain contains a dinucleotide-binding site that is capable tions in either the PXDLS-binding pocket in CtBPs or in of binding both NAD+ and nicotinamide adenine dinucleo- the binding motif of interacting proteins is sufficient to tide (NADH) (17). Although CtBPs are functional dehydroge- abrogate their interaction (2, 10–14). Moreover, this hydro- nases, the significance of this observation is not yet known, phobic cleft is essential to CtBP function because it plays and the physiological substrate for CtBP deacetylase activ- an extensive role in recruiting other members of the CtBP ity has not been identified (18). However, it has been estab- corepressor complex in both a PXDLS-dependent and a lished that the ability of CtBPs to bind NADH is required for PXDLS-independent manner (10). Specifically, the PXDLS- these proteins to form dimers with one another and exert binding site is essential for recruiting the core machinery transcriptional repression (10, 18–20). Nonetheless, CtBP of the corepressor complex, including histone deacety- mutants that are deficient in their ability to form dimers do lases (HDACs), histone methyltransferases (HMTases), display the ability to mediate other CtBP functions, which and transcriptional repressors, which are required for are discussed in further detail below (10).

Figure 1 Structures of CtBP1 and CtBP2. The PXDLS-binding cleft, RRT-binding cleft, and dehydrogenase domain of CtBP1 and CtBP2 compose the majority of the CtBP protein structure. Note the longer N-terminus of CtBP2, which contains an NLS that is absent from CtBP1. Also note that CtBP1 contains a PDZ domain at the C-terminus that is absent from CtBP2. T.R. Stankiewicz et al.: CtBPs in development and disease 491

Collectively, the PXDLS-binding cleft, RRT-binding cleft, and dehydrogenase domain of CtBP1 and CtBP2 compose the majority of the CtBP protein structure and are highly homologous for both CtBP family members and their various isoforms (Figure 1). However, there are also slight differences in protein sequence that contribute to the differing functions of CtBP1 and CtBP2. Most notably, CtBP2 contains a nuclear localization signal (NLS) at its N-terminus that is absent in CtBP1 (21). This region of CtBP2 contains several lysine residues that are acetylated, contributing to the nuclear retention of this protein (22). The NLS is absent in some isoforms of CtBP2, resulting in the accumulation of a small cytoplasmic pool of this protein; however, the cytoplasmic function of CtBP2 remains unclear. In contrast, CtBP1 relies on binding Figure 2 Transcriptional regulation by CtBPs. to transcriptional repressors such as zinc finger E-box pro- As CtBPs do not bind to DNA directly, they recruit various chromatin- teins (ZEB1/2), SUMOylation, and other interactions such as modifying enzymes to exert transcriptional repression. CtBPs can heterodimerization with CtBP2 for nuclear import and reten- recruit a corepressor complex consisting of HDAC1/2, Ubc9, and tion (21, 23, 24). Thus, CtBP1 can accumulate in the cyto- the HDAC1/CoREST/LSD1 complex. CtBPs can also recruit an HMT plasm where it mediates membrane trafficking and Golgi complex comprised of G9a, WIZ, CDYL, and GLP, and the SUMO E3 fission during mitosis (25, 26). Moreover, CtBP1 possesses a ligases, HPC2 and PIAS1, that repress gene transcription through histone modifications and/or by acting as a stage for the SUMOyla- PDZ-binding domain at its C-terminus that is not present in tion of a number of CtIPs. CtBP2, allowing CtBP1 to interact with proteins such as neuronal nitric oxide synthase (nNOS) to maintain its cytoplas- PXDLS-binding cleft and the RRT-binding cleft located mic localization (23). Other structural differences in CtBPs near the dinucleotide-binding site for NADH (10, 16). are the result of either alternative splicing or transcriptional Additionally, the PXDLS-binding site may bind multiple initiation from alternative promoter regions that produce the proteins at once, one containing a PXDLS-binding motif various isoforms of CtBP1 and CtBP2. and others interacting through PXDLS-independent asso- ciations (10). Therefore, CtBPs may mediate a diverse number of histone remodeling reactions that ultimately CtBP corepressor activity result in transcriptional silencing of a target gene. Further, as NADH binding is essential for the formation of CtBP The primary function of CtBP family members is transcrip- dimers, it is also necessary for the corepressor activity of tional repression. Because CtBPs do not possess intrinsic these proteins. Moreover, NADH binding to CtBPs affects DNA-binding capabilities, these proteins are recruited to the overall stability of CtBP-binding interactions, enhanc- active transcription sites by DNA-binding transcriptional ing the affinity of CtBPs for binding to interacting partners repressors such as ZEB1/2, Krüppel-like factors, Elk4, and via the PXDLS-binding pocket (20). E2F7 (14, 24, 27–29). Once bound to the repressor, CtBPs A majority of the corepressor activity associated with recruit histone-modifying enzymes to effectively halt tran- CtBPs is mediated by the activity of HDACs, which may scriptional activity at sequence-specific gene promoters rec- interact directly with CtBPs or be recruited as part of the ognized by the transcriptional repressors (Figure 2). Among HDAC1/CoREST/LSD1 corepressor complex (10). These these enzymes are HDAC1/2, Ubc9, and the HDAC1/CoREST/ enzymes deacetylate histone 3 at lysine 9, and this site lysine-specific demethylase 1 (LSD1) complex, which com- is subsequently methylated by the activity of auxiliary prise the core machinery of the corepressor complex (10). HMTase components such as G9a (31). Subsequent to this There are also a number of proteins including an HMT reaction, LSD1, which depends on recruitment by Znf217 complex comprised of G9a, WIZ, CDYL, and GLP, and three and CoREST to interact with CtBP1, is able to demethylate SUMO E3 ligases, HPC2, PIAS1, and Pc2 that act as auxiliary lysine 4 located on histone 3 (32). Notably, histone 4 has components in the corepressor complex, enhancing gene also been shown to be a target of SUMOylation, and this repression through histone modifications and acting as a modification has been linked to transcriptional repression stage for the SUMOylation of a number of CtIPs (10, 30). (33). Thus, there is also speculation that the SUMOyla- Structurally, CtBPs are capable of binding members tion machinery associated with the corepressor complex, of the corepressor complex at both the hydrophobic including Pc2, Ubc9, HPC2, and PIAS1, also plays a role 492 T.R. Stankiewicz et al.: CtBPs in development and disease in modifying histones to silence target gene expression. (dCtBP) performs corepressor functions that are largely SUMOylation is also thought to modulate the activity of consistent with the function of mammalian CtBP homo- components of the corepressor complex to either enhance logues. However, a recent study suggested that dCtBP is or restrict their function, thereby regulating the extent to able to modulate Wingless signaling through both repres- which gene expression is repressed. This will be discussed sion and activation of the Wingless target genes (19, 44). in further detail in the following section. Although the precise mechanism by which this activation CtBPs can also mediate transcriptional repression occurs is not well characterized, evidence suggests that through a mechanism independent of the corepressor the role of dCtBP in determining if Wingless targets are complex. This occurs by CtBPs directly binding and activated or repressed is largely dependent on its dimeri- inhibiting the histone/factor acetyltransferase p300 and zation state (19). Whereas dCtBP dimers exert transcrip- the transcriptional activator P/CAF (15, 34). Intriguingly, tional repression in this context, dCtBP monomers are SUMOylation of p300 reverses its activity and causes it capable of stimulating transcription of Wingless targets to act as a transcriptional repressor (35). Therefore, it is (19). dCtBP also plays a putative role in the control of Dros- possible that recruitment of the SUMOylation machin- ophila clock genes and regulation of circadian rhythms as ery to this protein by interactions with CtBPs may also overexpression of this protein increases mRNA expression play a role in inhibiting the transactivating function of a number of clock genes in vivo. In vitro analysis of a of p300, contributing to transcriptional repression of potential mechanism for this regulation of clock genes target genes (36). revealed that dCtBP requires the presence of the transcriptional activator, CLOCK/CYCLE (45). This suggests that some transcriptional activation by dCtBPs may be Derivation and function of CtBP isoforms mediated by the association of these proteins with clas- sical transcriptional activators. Following the description Both the CtBP1 and CtBP2 genes produce multiple iso- of context specific transcriptional activation by CtBPs in forms. In addition to their nuclear functions, some of these Drosophila, similar activity was attributed to mammalian isoforms play important roles within the cytoplasm that CtBPs in relation to Rac, a member of the Ras superfamily are unrelated to CtBP corepressor activity. For example, of small GTPases that are central regulators of cell migra- the CtBP1 gene produces long and short isoforms known tion and oncogenesis, and the Rac-specific guanine nucle- as CtBP1-L and CtBP1-S, respectively (37). Although both otide exchange factor (GEF) Tiam1 (46, 47). Nonetheless, of these isoforms are capable of participating in tran- examples of transcriptional activation by CtBPs are rare scriptional corepression at the nucleus, they also possess and require further analysis before firm mechanistic con- unique cytoplasmic functions. Indeed, CtBP1 has been clusions on this activity can be made. found to localize to ribbon synapses in sensory neurons, where it is thought to provide structure to synaptic ribbons (38, 39). In addition, CtBP1-S, more commonly known as CtBP/BARS, is known to participate in the process of mem- Regulation of CtBPs brane trafficking and Golgi partitioning during mitosis (25, 26) as well as regulate membrane composition in cancer Post-translational modifications cells (40). In addition to CtBP1, the CtBP2 gene also produces two splice variants of CtBP2 known as CtBP2-L and Although CtBPs are regulated by a number of factors, post- CtBP2-S, as well as a third isoform known as RIBEYE, which translational modifications are the best characterized (21, functions predominantly in the cytoplasm at ribbon syn- 22, 48–52). For example, phosphorylation of CtBPs often apses and has been extensively characterized in sensory targets these proteins for ubiquitination and subsequent neurons and bipolar cells (37, 41–43). Nonetheless, while proteasomal degradation. In many cases, when degrada- the non-nuclear functions of CtBP isoforms are important, tion of CtBPs occurs under stress conditions, this leads they will not be discussed in further detail here. to cell death. Among the stress-activated kinases that phosphorylate CtBPs, homeodomain-interacting protein kinase 2 (HIPK2) and c-Jun N-terminal kinase (JNK) are CtBPs and transcriptional activation the best characterized (51, 52). Both of these proteins have been shown to phosphorylate CtBP1 at Ser422 in Recent studies suggest a novel function for CtBPs in response to damaging ultraviolet (UV) irradiation of transcriptional activation. For example, Drosophila CtBP cancer cells, but only HIPK2 has been demonstrated to T.R. Stankiewicz et al.: CtBPs in development and disease 493 also phosphorylate CtBP2 at the homologous site (Ser428) both corepressor and dehydrogenase activity in the pres- (51, 52). Phosphorylation at Ser422 results in enhanced ence of NADH, following phosphorylation by PAK1. Thus, ubiquitination of CtBP1 and proteasomal degradation, although both AMPK and PAK1 suppress CtBP function, leading to derepression of CtBP target genes (Figure 3A). the fate of CtBP may be determined in a context-specific It has also been demonstrated that CtBP1 can be phospho- manner, with cellular stress inducing CtBP degradation rylated by AMP-activated protein kinase (AMPK) at Ser158 and subsequent apoptosis, whereas CtBP phosphoyla- in response to metabolic stress such as glucose with- tion stimulates downstream of mitogenic factors, which drawal (49). Similar to phosphorylation of Ser422 by JNK promote transient suppression of CtBP function that may and HIPK2, phosphorylation at Ser158 by AMPK results be required for pro-survival signaling to occur (53). Lastly, in ubiquitination of CtBP1 and degradation, thus inhib- CtBP1 can also be phosphorylated and targeted for pro- iting CtBP repressor function. Meanwhile, p21-activated teasomal degradation by Akt at Thr176, a reaction that is kinase 1 (PAK1) also phosphorylates CtBP1 at Ser158, but facilitated by interactions of Pc2 with both CtBP1 and Akt intriguingly, it does not trigger ubiquitination or degrada- (50). This interaction causes these proteins to localize to tion of this corepressor, although phosphorylation at this polycomb groups, which are known to function in tran- site appears to suppress the corepressor activity of CtBP1 scriptional repression. The effect of phosphorylation on directly (48). This occurs via a transient loss of nuclear CtBP expression and stability is summarized in Figure 3. CtBP localization, which occurs in conjunction with a con- The polycomb protein, Pc2, also plays another role in formational change in CtBP1 structure resulting in loss of the regulation of CtBP1 by acting as a SUMO E3 ligase for

Figure 3 Regulation of CtBPs. (A) Under conditions of cellular stress, CtBPs are phosphorylated by a number of kinases (e.g., JNK, Akt) that target CtBPs for ubiquitin- dependent proteasomal degradation. In addition to kinases, ARF interacts with CtBP2 through a hydrophobic region within the ARF protein, and this interaction leads to proteasomal degradation of CtBP2 in a phosphorylation- and ubiquitin-independent manner. Finally, CtBPs are stabilized by a PXDLS-dependent interaction with Bcl-3, a pro-survival member of the Bcl-2 family of proteins. (B) Under 5K apoptosis conditions, CtBPs are downregulated in neurons undergoing apoptosis in a mechanism that is dependent on intact miRNA biomachinery. 494 T.R. Stankiewicz et al.: CtBPs in development and disease this protein (30). Although CtBP2 can also be SUMOylated a number of proteins within the complex contain SUMO- by the same factors that SUMOylate CtBP1, this process interacting motifs (SIMs), which suggests that SUMOyla- occurs to a much lesser extent, and its effect on CtBP2 tion could play a role in the recruitment of complex activity is not currently understood (30). SUMOylation members, and in maintaining the overall stability of the has been shown to regulate a number of corepressors and corepressor complex over time (54). their associated complexes, and SUMOylation of CtBP1 CtBP1 and CtBP2 are nearly identical in their domain in particular, appears to regulate the localization of this structure and function; however, CtBP2 possesses an protein within the cell (23, 54). CtBP1 is SUMOylated at additional 20 amino acids at its N-terminus that play a Lys428, leading to retention of CtBP1 in the nucleus. significant role in regulating its function. In particular, This modification of CtBP1 directly opposes interactions CtBP2 possesses three lysine residues at positions 6, 8, with proteins such as nNOS, which enhance cytoplasmic and 10, all of which can be acetylated by the transcrip- localization of CtBP1 (23). Indeed, mutants lacking this tional activator p300 (22). Although acetylation does residue in the SUMOylation consensus sequence have a occur at all three residues, acetylation at Lys10 is particu- largely cytoplasmic distribution and this correlates with larly required for nuclear retention of CtBP2 (22). It is also decreased corepressor activity. noteworthy that CtBP activity can be regulated by acet- Several members of the SUMOylation machinery are ylation of CtIPs such as E1A and RIP140. In both cases, known interacting partners of CtBP1. In particular, Ubc9 acetylation of lysine residues flanking the PXDLS-bind- was identified as part of the core corepressor machin- ing motif located in these proteins results in decreased ery that associates with and SUMOylates CtBP1 (10, 30). CtBP association and reduced transcriptional repression Interestingly, although Ubc9 is sufficient to SUMOylate (58–60). Thus, CtBP activity can be regulated indirectly CtBP1 in vitro, it has also been found that this reaction is by enhancing or inhibiting its ability to bind CtBP-inter- facilitated by Pc2. Indeed, Pc2 interacts with both CtBP1 acting partners. and Ubc9 as an E3 SUMO ligase to enhance the repressor activity of CtBP1 (30). Moreover, the interaction between CtBP1 and the SUMOylation machinery during transcrip- Metabolic and redox sensitivity of CtBPs tional repression may act as a SUMOylation center for other members of the corepressor complex as a number of Similar to 2D hydroxyacid dehydrogenases, CtBPs contain these factors are known to be regulated by SUMOylation a Rossman fold that is necessary for NADH binding. Thus, (10, 54). In particular, HDAC1 is modified by SUMO, which there has been speculation that these proteins may alter increases its HDAC activity and enhances transcriptional their activity based on the metabolic and redox state of the repression (55). Moreover, SUMOylation of this protein cell. Although both forms of this dinucleotide are capable regulates its association with different members of the of inducing CtBP dimerization, CtBPs bind NADH with corepressor complex, decreasing its interaction with up to 100-fold higher affinity than NAD+, suggesting that CoREST, and increasing its association with ZNF198, these proteins are in fact sensitive to the ratio of NADH to another member of the CoREST complex that associ- NAD+ within the nucleus (18, 61, 62). Nonetheless, addi- ates with CtBP (56). This illustrates that SUMOylation tional reports have suggested that CtBPs bind both NAD+ not only regulates the activity of various components of and NADH with similar affinity (17, 63). Therefore, further the corepressor complex, but it also may regulate their investigation is required to determine which binding para- localization within the complex to decrease or enhance digm is correct. transcriptional repression. Other members of the core- Although CtBPs function as dehydrogenases, NADH pressor complex that can be SUMOylated include LSD-1, regulation is more likely involved in increasing the sta- CoREST, Znf198, and Pc2 (56). ZEB1, a repressor that is bility of CtBP dimers rather than stimulating any cata- known to mediate its activity through the CtBP corepres- lytic activity (64). This is most clearly demonstrated sor complex, is also SUMOylated. ZEB1 SUMOylation has by mutant forms of CtBPs lacking either the capacity been suggested to disrupt interactions with the corepres- to bind NADH or dehydrogenase activity. Whereas the sor complex (10); however, this has not been demon- latter mutants are capable of mediating corepressor strated experimentally, and previous evidence suggests activity, CtBPs deficient in dinucleotide-binding capac- that SUMOylation of ZEB1 is required for this protein to ity are expressed at a lower level, suggesting they are achieve its full function as a transcriptional repressor inherently less stable and are functionally impaired in (57). Although the effect of SUMOylation is not clear for terms of corepressor activity (64). This is contrary to all members of the corepressor complex, it is notable that a previous report demonstrating that CtBP2 mutants T.R. Stankiewicz et al.: CtBPs in development and disease 495 lacking dehydogenase activity display corresponding Bcl-3 (Figure 3A). Bcl-3 protein interacts with CtBP1 in a decreases in corepressor activity (20). These oppos- PXDLS-dependent manner to stabilize CtBP1 expression ing observations could be reconciled by findings in a within the cell (66). This process is mediated by blocking recent study suggesting that some catalytic mutants of CtBP1 proteasomal degradation and results in suppres- CtBP also display reduced binding affinity for NADH, sion of pro-apoptotic genes (66). although these mutants are still capable of forming CtBP In addition, recent evidence also demonstrates that dimers (63). Furthermore, a study by Madison et al. (63) CtBPs can also be regulated by the tumor suppressor alter- recently proposed that CtBPs not only form functional native reading frame (ARF), which interacts with CtBP2 dimers, but also higher-order tetramers that are also through a hydrophobic region within the ARF protein essential to CtBP function and are controlled by a tryp- located between residues 46 and 51, and this interaction tophan residue (Trp318) that acts as dimerization switch leads to proteasomal degradation of CtBP2 and cell death following binding to NADH. (Figure 3A). The mechanism by which ARF represses CtBP Consistent with this idea, NADH binding has been expression is presently unknown as ARF does not appear previously shown to conformationally alter CtBP to adopt to cause the phosphorylation of CtBP2 nor does it increase a ‘closed’ state that is more amenable to CtBP oligomeri- CtBP2 ubiquitination beyond basal levels, both of which zation (17). Moreover, increasing concentrations of NADH target CtBP to the proteasome (67). have been shown to increase interactions with other Finally, CtBPs may also be regulated by their localiza- proteins containing a PXDLS-binding motif by inducing tion to specific areas of the cell, or even to specific proteins oligomerization, enhancing CtBP binding to transcrip- by other CtBP-interacting partners. For instance, CtIP has tional repressors, and increasing transcriptional repres- been demonstrated to recruit CtBPs to proteins such as sion both in vitro and in vivo (20, 62). Collectively, these oncogenic proteins Rb and BRCA (68, 69), although CtIP results suggest that CtBPs are indeed sensitive to the does not always require CtBP recruitment to mediate tran- NAD+/NADH ratio, and by extension, the metabolic and scriptional repression (70–72). Thus, several studies have redox state of the cell. Given that the redox balance and demonstrated that CtBPs are regulated by many signaling metabolic state of the cell is significantly affected by a pathways that are involved in cellular survival and death. variety of cellular events, including growth, oncogenesis, and apoptosis, it is likely that the NAD+/NADH ratio plays an important role in determining the functions of CtBPs in these processes. However, a direct correlation between CtBPs in development the metabolic and redox state of the cells and CtBP activity has not been definitively established in these processes Invertebrates and non-mammalian models and warrants further investigation. Given the diverse and critical functions of CtBPs, it is perhaps not surprising that these proteins have impor- Other regulators of CtBPs tant functions during development. Indeed, mRNA transcripts of CtBPs are widely expressed (6, 73–75). Further, Whereas post-translational modifications and NADH zygotic mutations of dCtBP are embryonic lethal, suggest- binding are currently the best characterized modes of reg- ing an important involvement of dCtBP in development ulating CtBP activity, other protein regulators are begin- (76–78). The involvement of dCtBP during embryonic ning to emerge. As a result, the role of CtBPs in a number development is consistent with its role in transcriptional of cellular processes is expanding, revealing these pro- repression as CtBPs are recruited by short-range repres- teins to be the target of many signaling pathways involved sors such as Snail, Knirps, and Krüppel (79, 80). In addi- in cell death and survival as well as onocogenesis. Indeed, tion to functioning during Drosophila development, a recent study revealed that caspases indirectly mediate CtBPs also regulate Xenopus embryonic development, the downregulation of CtBPs in neurons under some cellu- as expression of an xCtBP fusion protein designed to lar conditions as removal of depolarizing potassium from enhance CtBP transcription resulted in loss of head, eyes, cerebellar granule neuron (CGN) cultures (5K apoptotic and shortened anterior-posterior axes (81). Although conditions) was toxic and resulted in reduced expression CtBPs have essential functions during development, rela- of CtBP1 and CtBP2 in a caspase- and micro-RNA (miRNA)- tively few studies have examined the precise involvement dependent manner (Figure 3B) (65). In addition to cas- of CtBPs in central nervous system (CNS) development or pases, CtBPs are also regulated by the pro-survival protein neuronal survival. 496 T.R. Stankiewicz et al.: CtBPs in development and disease

CtBPs in avian and murine development earlier embryonic death at E9.25 (6). These findings demonstrate that CtBP1 and CtBP2 exhibit both unique and During avian development, although CtBP1 and CtBP2 are redundant functions during mammalian development. often expressed in the developing embryo in overlapping This study described above examining CtBPs during domains, CtBPs also exhibit region- and temporal-specific murine development is consistent with normal human expression patterns. For instance, although only CtBP2 is tissues and human cancer cell lines, which also dem- expressed in the primitive streak during early develop- onstrate overlapping and unique expression of CtBP1 ment, both CtBP transcripts are expressed in this domain and CtBP2 (75). Nonetheless, expression of CtBP2 differs at later developmental stages (82). These findings are in between humans and mice, particularly in skeletal muscle agreement with murine studies that also demonstrate where CtBP2 expression is greater in humans (73). There- duplicative and unique roles for CtBP1 and CtBP2 during fore, in future studies, it will be important to dissect the development and CNS maturation. For instance, although functional significance of unique and overlapping pat- CtBP1 homozygous null mice are small and show a 23% terns of CtBP1 and CtBP2 expression during development reduction in viability at P20, those that do survive are and CNS maturation. fertile (Figure 4) (6). In contrast, CtBP2 homozygous null mice fail to develop beyond E10.5 and demonstrate axial truncations and delayed development of the forebrain CtBPs and CNS development and hindbrain (6). In support of overlapping functions for CtBP1 and CtBP2, the generation of various compound To date, the involvement of CtBPs in the nervous system mutant mice resulted in dosage-sensitive developmental has mostly been limited to the role of these proteins in defects. For example, reducing the expression of CtBP2 development. For example, dCtBP negatively regulates in CtBP1-/- mice exacerbated the CtBP1-/- phenotype as the formation of mechanosensory bristles as loss-of- CtBP1-/- CtBP2+/- mice died in utero and exhibited increased function dCtBP mutants exhibit supernumerary mecha- developmental defects and abnormalities in the formation nosensory bristles, whereas gain-of-function dCtBP of several skeletal structures, such as failure to generate mutants demonstrate a marked loss of bristles (78, 83). cartilaginous and fully ossified skeletal elements in the dCtBP may influence the appearance of ectopic bristles ribs and vertebrae. In a similar manner, CtBP1+/- CtBP2-/- through extrasensory organ precursor cells, which are mice also demonstrated enhanced developmental defects also increased in loss-of-function dCtBP mutants (78). In such as failure to complete neural tube closure. This is a addition to regulating the formation of mechanosensory marked difference from CtBP1+/+ CtBP2-/- mice, which com- bristles, dCtBP also promotes eye and antennal specifi- pleted neural tube closure. Finally, mice homozygous null cation through interactions with Eyeless, Distal antenna, for both CtBP1 and CtBP2 demonstrated the most severe Distal antenna related, and Daschund proteins (27). embryonic phenotype displaying minimal heart morpho- Moreover, CtBP1 and CtBP2 also differentially mediate genesis, blebs and blisters in the neuroectoderm, and nervous system development in the developing chick.

Figure 4 CtBPs in development. (A) Wild-type mouse embryo. (B) CtBP1-/- knockout mouse. Note that CtBP1-/- knockout mice are small and viable but show a reduced survival rate. (C) CtBP2-/- knockout mouse. Note that CtBP2-/- knockout is embryonic lethal, and these mice show greater developmental defects when compared with CtBP1-/- knockout mice. (D) CtBP1-/-/CtBP2-/- knockout mouse. Note that CtBP1/CtBP2-null mice demonstrate enhanced developmental defects and earlier embryonic death when compared with either CtBP1-/-/CtBP2+/+ or CtBP1+/+/CtBP2-/- mice. E, embryonic day. T.R. Stankiewicz et al.: CtBPs in development and disease 497

For instance, emerging neural crest cells express CtBP2, absent from the alternative splice variant CtBP2-S, which whereas dorsal root ganglia express CtBP1 (84). The appears to be the major CtBP2 isoform localized to syn- importance of CtBP expression during avian CNS devel- apses. In contrast, CtBP2-L contains the NLS and localizes opment is highlighted by the key involvement of these more exclusively to neuronal nuclei (86). The localization proteins in regulating the transition of neural precursor of CtBP1 and CtBP2 to pre-synaptic terminals may indi- cells in the ventricular zone of the dorsal spinal cord cate a function for CtBPs in neurotransmitter release as from a proliferative to a differentiated state (85). Further- CtBP1 has been implicated in both membrane fission and more, as previously mentioned, CtBPs appear to have an fusion (87). Furthermore, RIBEYE, is the main component important role in neural tube closure and development and scaffold for ribbon synapses in sensory neurons such of the forebrain and hindbrain in mice (6). These previ- as photoreceptor and hair cells, suggesting an important ous reports highlight critical functions for CtBPs during function for RIBEYE in fine-tuning the tight release of syn- development and specifically suggest an essential role for aptic vesicles required to detect a wide range of stimulus CtBPs in neuronal development. intensities important for proper vision and hearing (38). These previous reports suggest several important functions for CtBPs in the adult brain, which are both con- Other functions of CtBPs in the nervous sistent with and distinct from their well described role in system transcriptional repression.

In addition to neuronal development, recent reports indicate that CtBPs may critically mediate other aspects of neuronal function in the adult murine brain. For instance, CtBPs in apoptosis although the significance remains to be elucidated, nuclear expression of CtBP2 was higher in excitatory cells Consistent with their major roles in transcriptional core- when compared with inhibitory cells of the hippocam- pression, early studies demonstrated that CtBPs promote pus, whereas CtBP1 demonstrated higher nuclear expres- cellular survival primarily through repression of pro-apo- sion in inhibitory cells (86). Moreover, although previous ptotic molecules of the Bcl-2 family of proteins (Figure 5). evidence suggests that CtBP1 functions principally in the For instance, mouse embryonic fibroblasts (MEFs) defi- nucleus, CtBP1 expression has also been demonstrated in cient in both CtBP1 and CtBP2 expression were hypersen- pre-synaptic terminals of cultured hippocampal neurons, sitized to apoptosis initiated by diverse stimuli such as Fas highlighting a potential function for CtBP1 in learning and ligand, staurosporine, and UV irradiation. Furthermore, memory that is distinct from its role as a transcriptional increased expression of the pro-apoptotic molecules corepressor (38). In agreement with a pre-synaptic func- PERP, Bax, Bik, Puma, p21, and Noxa was observed in tion for CtBPs, CtBP2 also colocalized with the pre-syn- CtBP-deficient MEFs (Figure 5) (7, 88, 89), and enhanced aptic marker Bassoon in the cerebellum, and to a lesser expression of both PERP and Bax was rescued by intro- extent, the hippocampus and cerebral cortex. The NLS is duction of CtBP1 (Figure 5) (7, 88). Indeed, expression

Figure 5 CtBPs as repressors of apoptosis and anoikis gene programming. CtBPs promote cellular survival through transcriptional repression of the pro-apoptotic molecules Bax, Bik, Puma, Noxa, PERP, p21, Bim, and Bmf. CtBPs also repress epithelial gene programming associated with anoikis insensitivity by repression of E-cadherin, occludin, plakoglobin, and keratin 8. 498 T.R. Stankiewicz et al.: CtBPs in development and disease of active caspase 3, the death executioner of apoptosis, CtBPs and neuronal survival and its cleaved substrate, poly(ADP-ribose) polymerase (PARP), were reported in CtBP-null MEFs exposed to UV Although largely unexplored, the essential involvement radiation (90). Furthermore, osteosarcoma cells subjected of CtBPs in mediating survival in non-neuronal cells and to siRNA knockdown of CtBP2 demonstrated enhanced their central roles in nervous system development high- expression of the pro-apoptotic Bcl-3 homology 3 domain lights a potential function for CtBPs in regulating neu- (BH3)-only proteins Bim and Bmf (89). It is important ronal survival. Indeed, we recently reported that CtBPs to note that transcriptional repression of pro-apoptotic undergo caspase-dependent downregulation in primary genes does not necessarily require the dehydrogenase CGNs exposed to a variety of neurotoxic insults, including activity of CtBP, as mutants defective in dehydrogenase removal of depolarizing potassium (5K apoptotic condi- activity inhibited apoptosis to a similar degree as wild- tions), incubation with the BH3-only mimetic HA14-1, the type CtBP (7). nitric oxide donor, sodium nitroprusside, or the complex I inhibitor, 1-methyl-4-phenylpyridinium. In addition, CtBPs also undergo downregulation in N27 dopaminergic CtBPs and p53-independent pathways cells exposed to 6-hydroxydopamine. Further establish- ing an essential pro-survival function for CtBPs in CGNs, Although most, if not all, of the aforementioned genes either antisense mediated downregulation of CtBP1 or that are repressed by CtBPs are transcriptional targets treatment with the CtBP inhibitor, 4-methylthio-2-oxobu- of the pro-apoptotic molecule p53, loss of CtBP function tryic acid (MTOB), induced significant neuronal apoptosis appears to induce apoptosis through a p53-independent concomitant with an increase in the pro-apoptotic BH3- mechanism in many cell systems. For example, siRNA only protein Noxa (65). Intriguingly, although the degra- against CtBPs or genetic knockout of CtBPs resulted in dation of CtBP1 occurred in a caspase-dependent manner caspase activation and subsequent apoptosis in p53-defi- in CGNs exposed to 5K apoptotic conditions, the kinetics cient H1299 cells (90). CtBPs also interact with a BTB of CtBP1 degradation were not enhanced and recombinant domain-containing protein, CZBTB38, in p53 knockout CtBP1 was not cleaved in vitro by caspase 3. Furthermore, MEFs to enhance caspase-3 activation and apoptosis (91). 5K apoptotic conditions did not have a significant effect Collectively, these data demonstrate that degradation of on CtBP transcript expression. However, mouse embry- CtBPs can induce apoptosis by a mechanism that is not onic stem cells displayed caspase-dependent downregu- dependent on the tumor suppressor p53. lation of CtBP1 following exposure to staurosporine, an In UV radiation-induced apoptosis, HIPK2 has effect that was not observed in DGCR8 knockout cells that emerged as a key regulator of CtBP downregulation. are deficient in miRNA processing. Therefore, caspases Although HIPK2 typically induces apoptosis in response appear to regulate the expression of CtBP indirectly at a to UV radiation by phosphorylating and activating p53, post-transcriptional level via a mechanism that is depend- HIPK2 can also induce cell death in a p53-independent ent upon miRNA biomachinery (Figure 3B). These data manner through phosphorylation of Ser422 and subse- demonstrate that CtBPs can undergo caspase-dependent quent proteasomal degradation of CtBP1. These results downregulation in neurons undergoing apoptosis as an are also consistent with previous reports that HIPK2 can alternative mechanism to proteasomal degradation, as signal cell death either through p53 or in a p53-independ- incubation with the proteasome inhibitor MG132 did not ent manner through the activation of JNK. Intriguingly, prevent the downregulation of CtBPs in CGNs exposed either UV radiation or JNK activation resulted in phos- to 5K apoptotic conditions. This report identifies a previ- phorylation of CtBP1 on Ser422, proteasomal degrada- ously undescribed role for CtBPs in maintaining the sur- tion, and apoptosis of human lung cancer cells (51). The vival of primary neurons and further underscores that the effector function of HIPK2 on JNK may be modulated downregulation of CtBPs can be triggered in neurons by through SUMOylation as human HIPK2 is SUMOylated in exposure to a variety of neurotoxic insults that have previ- vitro and this modification inhibited HIPK2-dependent ously been implicated in neurodegenerative disease. JNK activation and subsequent apoptosis of p53-deficient Hep3B hepatoma cells (92). Although downregulation of CtBPs induces apoptosis in cells lacking p53, experimen- CtBPs and Rac GTPase tal evidence also demonstrates that CtBP may regulate the expression of p53 under certain cellular conditions in Intriguingly, we have also reported a significant loss cancer cells (93, 94). of CtBPs during CGN apoptosis induced by the small T.R. Stankiewicz et al.: CtBPs in development and disease 499

GTPase inhibitors, Clostridium difficile Toxin B (ToxB), is localized to the nucleus. Intriguingly, Htt contains a and Clostridium sordellii lethal toxin (LTox) (65). As ToxB canonical PXLDS CtBP-binding site, suggesting that Htt (an inhibitor of Rac, Rho, and Cdc42) and LTox (an inhibi- may be involved in transcriptional repression. In human tor of Rac, Ras, and Rap) have overlapping specificity for fibroblasts, full-length wild-type Htt interacts with CtBP inhibiting Rac GTPase, these findings suggest that Rac in the nucleus to cause constitutive repression, and while GTPase may function upstream of CtBPs to regulate their a polyglutamine expansion in Htt impaired this interac- expression and consequently, the transcriptional repres- tion, mHtt that was targeted to DNA remained effective sion of BH3-only pro-apoptotic proteins, such as Noxa at transcriptional repression (95). Thus, mHtt may have (Figure 3). Although the precise mechanism by which a weaker interaction with CtBPs when compared with CtBPs are downregulated in neurons exposed to small wild-type Htt, leading to instability of protein complexes GTPase inhibitors requires further investigation, recent required for CtBP-dependent transcriptional repression of studies have suggested a positive correlation between pro-apoptotic genes. the expression of CtBPs and the activity of Rho family In addition to the potential involvement of dysregu- GTPases in non-neuronal cells. For example, overexpres- lated CtBP function as a factor underlying the progression of CtBP2 paradoxically enhances the transcription sion of HD, recent studies have also begun to elucidate of the Rac GEF Tiam1 and subsequently enhances the the involvement of CtBPs in regulating neuronal degen- Rac-dependent migration of human non-small cell lung eration following neuronal trauma. Following traumatic carcinoma cells (46). In conjunction with our own find- brain injury, both HIPK2 and CtBP2 were increased in the ings, these data suggest that CtBP1/CtBP2 and Rac GTPase peritrauma brain cortex when compared with contralat- may function in a positive feedback loop to regulate the eral cerebral cortex. However, while HIPK2 was associ- expression and activities of one another. Nonetheless, it ated with activation of caspases and neuronal apoptosis, is important to consider that our studies demonstrating enhanced CtBP2 expression was associated with activation that CtBPs are regulated downstream of Rac GTPase were and proliferation of astrocytes (96). In contrast, CtBP2 was conducted in primary neurons, while the CtBP2-depend- downregulated in Schwann cells following sciatic nerve ent upregulation of Tiam1 and subsequent activation of crush in rats (97). Thus, these studies indicate that CtBP2 Rac GTPase was shown in H1299 cell lung and HCT116 may play a role in regeneration following peripheral nerve colon carcinoma cells. Thus, whether or not Rac GTPase injury. Nonetheless, the involvement of CtBPs in neuronal lies upstream or downstream of CtBP1 and CtBP2 may trauma are only beginning to be understood and further simply depend on the cell type. In future studies, it will be research is necessary to determine the precise involve- important to determine whether CtBP2 similarly triggers ment of CtBPs in traumatic nerve injury and regeneration. Tiam1-dependent activation of Rac GTPase in neurons and whether this has an effect on neuronal survival. Indeed, the link between CtBPs and Rho family GTPases is only CtBPs and neuroinflammation recently being elucidated and future studies will be necessary to decipher the precise relationship between CtBPs Intriguingly, CtBPs may also have important functions and Rho family GTPases in neuronal survival. in maintaining neuronal survival through their role in regulating the inflammatory response by microglia and astrocytes. In a recent study by Saijo et al. (98), it was CtBPs in Huntington disease and traumatic demonstrated that the endogenous estrogen receptor β nerve injury ligand, 5-androsten-3β,17β-diol (ADIOL), mediates the recruitment of CtBP1 and CtBP2 to the promoter region of Although not directly examined in human neurodegen- c-Jun/c-Fos AP1-dependent promoters, leading to the tran- erative disorders, previous in vitro and in vivo animal scriptional repression of genes that are implicated in acti- studies have linked dysregulated CtBP activity to the pro- vation of the inflammatory response in Th17 T cells. The gression of various neurodegenerative diseases, such as recruitment of CtBPs to DNA was dependent upon ERβ, Huntington disease (HD). HD is an autosomal dominant but not 17β-estradiol, as synthetic ERβ-specific ligands neurodegenerative disorder caused by inheritance of one promoted CtBP recruitment and prevented inflammation. mutant huntingtin (Htt) gene. Mutant Htt (mHtt) contains Indeed, the ERβ-specific ligands, indazole-Br and inda- an expanded polyglutamine repeat near the N-terminus zole-Cl, induced the interaction between ERβ and CtBPs and cleavage of mHtt results in an N-terminal fragment and inhibited the inflammatory response in microglia and that is toxic to medium spiny striatal neurons when it astrocytes. 500 T.R. Stankiewicz et al.: CtBPs in development and disease

In contrast to this prior study suggesting that CtBPs acquisition of anoikis insensitivity (Figure 5) (4). As are important molecules that exert transcriptional repres- epithelial cells contain prominent cell-cell and cell- sion of pro-inflammatory genes, a recent study indicates matrix adhesions, repression of epithelial genes such as that CtBPs may also function to induce the inflamma- E-cadherin is an important process in acquiring anoikis tory response in vivo in a rat model of neuroinflamma- insensitivity, allowing cells to survive following detach- tion. Indeed, CtBP2 expression was induced in activated ment from the ECM (100). MEF cells derived from CtBP1/2 microglia and astrocytes following lipopolysaccaride double knockout mice displayed enhanced expression of (LPS) administration to rats. Enhanced CtBP2 expression several epithelial-specific proteins, such as E-cadherin, may contribute to LPS-induced inflammation as CtBP2 plakoglobin, occludin, and keratin 8 (Figure 5) (7). In a knockdown in cultured microglia prevented the release CtBP1 rescue experiment, these effects were abrogated, of tumor necrosis factor α (99). Thus, the precise involve- underscoring an essential function for CtBPs in sup- ment of CtBPs in regulating the inflammatory response pressing epithelial genes. The ability of CtBP to repress likely varies depending on the expression of various toll- epithelial genes did not require dehydrogenase activ- like receptors as well as ERβ receptors on microglia cells ity as a glycine 183 to alanine mutation did not attenu- and astrocytes. Nonetheless, these findings are particu- ate the ability of CtBP1 to downregulate epithelial gene larly interesting given the critical involvement of aberrant expression (4). However, it has since been proposed that activation of microglia and astrocytes in the CNS during this particular mutation may only partially disrupt the the progression of multiple neurodegenerative diseases NADH-binding motif (105). In accordance with a function such as amyotrophic lateral sclerosis (ALS) and Parkinson of CtBPs in conferring anoikis insensitivity, MEFs defi- disease (PD). Therefore, future studies should be aimed cient in the expression of both CtBP1 and CtBP2 displayed at clarifying the involvement of CtBPs in regulating the enhanced sensitivity to detachment-induced anoikis inflammatory response of microglia and astrocytes. Given as evidenced by increased nuclear fragmentation and their pro-survival function in primary neurons and the caspase activation when compared with attached cells, potential to repress inflammation, CtBPs may represent a both effects of which were attenuated by reintroduction novel therapeutic target for the treatment of various neu- of CtBP1 (4). Collectively, these data highlight CtBPs as rodegenerative diseases. regulators of anoikis and suggest that dysregulation of CtBPs may underlie the metastatic nature of anoikis- insensitive tumor cells. CtBPs in anoikis

The term ‘anoikis’ refers to a specific type of apoptotic CtBPs and cancer death that occurs in response to loss of cell adhesion (100). This process is mediated by integrins that transmit The original identification of CtBPs as binding partners for intracellular signals in response to mechanical forces due E1A adenovirus suggested the involvement of CtBPs in reg- to contact with the extracellular matrix (ECM). It is impor- ulating oncogenesis. Indeed, an early study identified that tant for an organism to be able to remove displaced cells E1A mutants defective in their PXDLS domain enhanced to prevent their reattachment to new matrices. Despite a transformation in conjunction with the Ras oncogene in unique definition, anoikis is an apoptotic process that can rodent epithelial cells, suggesting that E1A may promote occur via either extrinsic or intrinsic apoptotic cascades oncogenesis via inhibition of CtBPs. More recently, a (101, 102). Anoikis that is executed through the intrinsic genome-wide analysis was performed to provide a more apoptotic cascade is largely carried out by the pro-apop- comprehensive description of CtBP repression targets. totic BH3-only molecule Bim, and to a lesser extent, Bid From this study, CtBP targets could be categorized into (103). Following detachment from the ECM, extrinsic three main categories: genes that regulate cell renewal apoptosis can also be activated via increased expression and pluripotency, genes that regulate genome stability, of Fas and Fas ligand (104). Both the intrinsic and the and genes that regulate epithelial differentiation and extrinsic anoikis pathways converge on activation of the prevent epithelial-to-mesenchymal transition (EMT) (40, death executioner of apoptosis, caspase 3. 106). Remarkably, these three classes also describe path- Evidence suggests that CtBPs are important modula- ways that are classically dysregulated in cancer. Indeed, tors of anoikis as enhanced activation of CtBPs represses many advances have been made in elucidating the role of the expression of epithelial genes in parallel with the CtBP in the pathogenesis of various cancers. T.R. Stankiewicz et al.: CtBPs in development and disease 501

CtBP regulation of tumor suppressor genes cells, resulting in the derepression of p53 (93). These findings are in contrast to the established paradigm that Phosphatase and tensin homologue (PTEN) is a known increased intracellular NADH levels promote CtBP inter- tumor suppressor that is involved in cell cycle regulation. actions with proteins containing a PXDLS-binding motif, It was discovered that overexpression of CtBP2 causes and indicate that the regulation of Hdm2/CtBP-mediated a decrease in PTEN expression levels and increases repression of p53 occurs via a distinct mechanism (20, 62), cell migration through activation of a PI3K-dependent consistent with a role in transcriptional repression, loss pathway (46). Furthermore, the Snail transcription factor, of CtBPs led to enhanced expression of p53; however, p53 which drives breast cancer metastasis, represses PTEN in exerted a pro-survival effect in cells deficient in CtBPs via a CtBP-dependent manner, which results in a pro-survival p53-dependent expression of p21WAF1, which has previ- effect (79, 107). ously been demonstrated to antagonize p53-dependent In addition to PTEN, other tumor suppressors, such cell death (94, 108). Thus, whether or not p53 is required as p53 and hypermethylated in cancer (HIC1), are modu- for cell death provoked by the downregulation of CtBPs lated by actions of CtBPs. Transcription of p53 target genes in cancer cells likely occurs in a cell type- and context- is negatively regulated through a direct physical interac- specific manner. In a similar manner to Hdm2, the tran- tion with the oncoprotein human double minute 2/mouse scriptional repressing qualities of the tumor suppressor double minute 2 (Hdm2/Mdm2). Hdm2/Mdm2 is a known protein HIC1 are dependent on its interaction with CtBP1 inhibitor of p53 that can inactivate this protein via mul- (11). Thus, CtBPs function as critical modulators of various tiple mechanisms, including proteasomal degradation, tumor suppressor proteins, whose regulation is consist- nuclear export, and reduced interaction with transcrip- ently implicated in the etiologies of multiple cancers tional coactivators. However, Hdm2 has also been demon- (Figure 6). Nonetheless, future studies will be critical to strated to recruit CtBP2 in a redox-sensitive manner to the further define the downstream effects of CtBP repression promoter region of p53 to exert transcriptional repression. of tumor suppressors and their putative causal involve- This interaction, which occurs through an acidic domain ment in the development of cancer. in Hdm2, is diminished under hypoxic conditions that Another important tumor suppressor that inter- increase the NADH/NAD+ ratio in MCF-7 breast cancer acts with CtBP is the adenomatous polyposis coli (APC)

Figure 6 Dysregulation of CtBPs as a common pathological link in neurodegenerative disease and cancer. CtBPs function to promote cellular survival through repression of pro-apoptotic (e.g., Bax, p53) and pro-anoikis (e.g., E-cadherin) gene programming. Typically, CtBPs suppress Wnt-dependent cell proliferation except in the case of mutant APC. CtBPs diminish DNA damage repair and cell cycle arrest. Finally, CtBPs enhance cell survival and migration through repression of PTEN. Thus, diminished expression of CtBPs may underlie aberrant neuronal apoptosis neurodegenerative disease, whereas enhanced CtBP expression may promote carcinogenesis by inhibition of apoptosis and stimulation of proliferation, migration, survival, and genomic instability. 502 T.R. Stankiewicz et al.: CtBPs in development and disease protein, which typically interacts with CtBP to repress future studies will be required to discern the importance Wnt target gene expression. APC prevents transcription of this interaction in known cancers. by targeting β-catenin for proteasomal destruction, pro- Mutations of ARF tumor suppressor are frequently moting nuclear export, or through sequestration. Impor- associated with hepatocellular carcinoma (HCC) (114). tantly, APC-mediated β-catenin sequestration relies on Although the causal mechanistic effects of these muta- APC interacting with CtBP, suggesting that these proteins tions remain to be determined, the implications for its may exist in a complex to mediate repression of Wnt- role in the development of cancer have yielded studies dependent gene transcription (109). Indeed, this associa- indicating a novel function of ARF in regulating CtBPs. tion has been demonstrated to be important in repression In vitro studies in lung carcinoma cells have revealed that of the c-myc oncoprotein (110). Mutations in APC disrupt exogenous expression of the tumor suppressor ARF has the interaction between APC and CtBP, a process that is a an antagonistic effect on CtBP2-mediated cell migration causal factor in aberrant Wnt signaling in cancer. Specifi- through directly targeting CtBP for proteasomal degrada- cally, APC loss-of-function mutations are observed in most tion (46, 67). Similarly, decreased ARF levels correlate with cases of colorectal cancer, including the familial adeno- increased CtBP levels in human colon adenocarcinoma, matous polyposis (FAP) syndrome. Furthermore, these further supporting that ARF may function as a repressor of interactions are also impaired in colorectal cancer cells CtBP (115). In contrast, ARF repression of tumor migration harboring mutations in APC, suggesting a causal rela- requires the interaction between ARF and CtBP in HCC cell tionship between APC-CtBP complex function and Wnt lines as well as pancreatic ductal adenocarcinoma models pathway signaling in cancer progression (Figure 6) (110). (116, 117). These data are interesting in the context of other CtBP also regulates the expression of several cyclin- studies that indicate repression of ARF may also occur dependent kinase inhibitors (CDKIs), known for their via a CtBP-dependent mechanism, demonstrating that a onco-suppressive properties through modulating cell complex feedback mechanism may be involved in CtBP/ cycle arrest. Specifically, increased CtBP expression tumor suppressor regulatory pathways (111). negatively regulates the tumor suppressor p16INK4a CtBP inhibition has also been demonstrated to occur and prevents cell senescence in human esophageal through the activity of the familial colon cancer-linked squamous cell carcinoma, suggesting that CtBP levels tumor suppressor APC, possibly through proteasomal may have a direct role in the evasion of cell cycle regu- degradation of CtBP (109, 118, 119). APC is shown to target lation in this tumor type (111, 112). Moreover, CtBP has CtBP for destruction, which is important in ameliorating been shown to repress expression of p21 (waf1/cip1) in CtBP suppression of intestinal differentiation through a PARP-dependent manner during DNA damage condi- retinol dehydrogenases (118). Accordingly, increased tions known to be prevalent in tumor micro-environ- CtBP1 levels are observed in tumors obtained from FAP ments (113). Although CtBP modulation of the cell cycle patients harboring mutations in APC, further implicating remains to be demonstrated in many tumor types, the APC in the repression of CtBP expression (118, 119). These implications of CtBP-dependent repression of CDKIs data are interesting in light of the previously described highlight a key role for CtBPs in a crucial upstream event involvement of APC and CtBP in regulating Wnt signaling. in the evasion of cell cycle arrest and tumor progression. The disparity may be due, in part, to cell type specificities The role of CtBPs in promoting cellular transformation in function. However, given the complex nature of CtBP through regulation of tumor suppressor genes is sum- regulation, it is also likely that a feedback mechanism marized in Figure 6. between CtBP and APC functions to regulate these two critical proteins. Collectively, these data also suggest that CtBP represents a convergence point in a complex mecha- CtBP repression by tumor suppressors nism involving APC/β-catenin, Wnt, and other factors such as retinol dehydrogenase, and that dysregulation of Although the role of CtBPs in repression of tumor suppres- this pathway due to mutations likely plays a significant sor genes is quite clear, in vitro studies also reveal that CtBP role in the development of colon adenocarcinomas. can be reciprocally repressed by the actions of other tumor suppressor proteins. As previously discussed, HIPK2 has been shown to mediate proteasomal degradation of CtBP Breast cancer/BRCA regulation through phosphorylation (51, 90), and reduced CtBP levels are associated with increased apoptosis, consistent with Breast cancer 1 and 2 (BRCA 1, 2) proteins are potent the tumor suppressive functions of HIPK2 (90). However, tumor suppressors that function in DNA repair. Mutations T.R. Stankiewicz et al.: CtBPs in development and disease 503 in these genes are consistently linked to several different (7). Importantly, CtBP recruitment to the E-cadherin pro- types of cancer, most commonly, breast cancer. As is the moter has been demonstrated to occur during hypoxic case with other tumor suppressor proteins, BRCA regu- conditions that are present in metabolically active tumor lation involves interactions with CtBP. Studies in head types (128). Moreover, CtBP overexpression has been cor- and neck squamous cell carcinomas reveal that BRCA1 related with decreased E-cadherin levels in human colon is repressed through CtBP1 binding to its promoter. Addi- cancers (129). Although CtBP is quite clearly involved in tionally, this interaction occurs in a redox-dependent the repression of E-cadherin, and therefore involved in manner and increases in hypoxic conditions associated EMT, modulation of CtBP recruitment to the E-cadherin with tumor micro-environments (120). Additional studies promoter is quite complex. Expression of E-cadherin is demonstrate that CtBP2 represses BRCA1 in ovarian cancer partially controlled through recruitment of CtBP by ZEB1 cell lines (121) and that loss of CtBP at the BRCA1 promoter and ZEB2 (7, 24, 130). Specifically, overexpression of results in increased BRCA1 expression (122). Finally, CtBP1 ZEB1 causes a decrease in E-cadherin in several cancers, is shown to repress BRCA2 expression through interac- including, breast cancer, uterine cancer, and colon tion with the slug repressor protein in human breast cancer (131–135). Interestingly, ZEB1 has also been dem- cancer cells (123). Interestingly, CtBP1 exists in a complex onstrated to dissociate from CtBP to form an activation with HDAC1 and p53 to inhibit BRCA2 transcription (124). complex with SMADs in response to tumor growth factor In contrast, studies in breast cancer-derived cell lines (TGF) β signaling, which is associated with enhanced demonstrate that CtBP exerts a repressive function on expression of mesenchymal-specific genes (130, 136). in p53-mediated transcription (94). Collectively, these Collectively, these data suggest that an increase in ZEB1 data describe a direct role for CtBP in the repression of levels likely causes an increase in CtBP recruitment and BRCA and the development of breast cancer, which has resultant repression of E-cadherin in various cancer proven to be a valuable mechanism in which to intervene types. Moreover, ZEB1 also enhances the expression of therapeutically. mesenchymal-specific genes in a CtBP-independent In contrast to data suggesting that CtBPs mediate manner downstream of TGF-β signaling. breast cancer progression via repression of BRCA genes, The regulation of cell adhesion proteins by CtBP is it has also been demonstrated that CtBP is negatively also a complex process. The cell adhesion-related pho- regulated in breast cancer. MUC1 is an oncoprotein that shoprotein, pinin/DRS (Pnn), has been shown to directly is overexpressed in some forms of breast cancer. Impor- interact with CtBP1 and interfere with its ability to repress tantly, this protein inhibits CtBP repression of cyclin D1 E-cadherin expression (137). CtBPs are also regulated at through interaction with TCF7L2, thus promoting cell the post-transcriptional level by the miRNA degradation cycle progression (125). Thus, the role of CtBPs in breast machinery. Specifically, miR-137 is shown to have tumor cancer, like other types of cancer, is complex and is likely suppressive properties in melanoma cells and increases regulated in cell type- and tumor micro-environment- E-cadherin levels through CtBP1 repression (138). specific contexts.

Other regulators of CtBP in cancer CtBP role in EMT The involvement of CtBP corepressor activity in colon Loss of cellular adhesion and acquisition of anoikis cancers and breast cancers is perhaps the most exten- resistance represents a critical step in the cellular tran- sively studied. However, CtBP transcriptional regulation sition to malignancy. This process involves a downreg- has been demonstrated in a variety of other cancers. ulation of epithelial-specific genes accompanied by an Upregulation of the ecotropic virus integration site 1 increase in expression of mesenchymal-specific genes, protein (Evi-1) is a hallmark of myeloid malignancies and allowing for a more motile and invasive mesenchymal myelodysplasia. Importantly, Evi-1 inhibition of TGF-β phenotype and is termed the ‘epithelial-to-mesenchymal pathway through repression of SMAD involves recruit- transition’ (126). EMT allows for a tumor cell’s motil- ment of CtBPs (139). Incidentally, SMAD repression may ity and is mediated through downregulation of cellular also occur through recruitment of CtBPs by ZEB (130). adhesion molecules such as E-cadherin (127) as well Increased expression and mislocalization of CtBP is as evasion of anoikis. Early studies demonstrated that evident in prostate cancer cells. Furthermore, knockdown several epithelial-specific genes are negatively regu- of CtBP in prostate cancer cell lines inhibited cell invasion lated by CtBP, including E-cadherin, and plakoglobin (140). In melanoma, CtBPs are shown to repress BRCA1 504 T.R. Stankiewicz et al.: CtBPs in development and disease and p16INK4a (141). Thus, dysregulation of CtBPs may be a common factor underlying the development of many different types of cancers.

Outlook

Targeting CtBPs in neurodegenerative disease

Recent evidence demonstrating that downregulation or inhibition of CtBPs is sufficient to trigger neuronal apoptosis underscores the potential therapeutic benefit of increasing the expression or activation of CtBPs as a therapeutic treatment option for neurodegenerative diseases (Figure 7A) (65). Indeed, this notion is supported by evidence suggesting that mHtt may have a weaker interaction with CtBPs when compared with wild-type Htt, leading to instability of protein complexes required for CtBP-dependent transcriptional repression and neu- Figure 7 CtBPs as novel therapeutic targets for the treatment of ronal survival (95). In future studies, it will be of critical neurodegenerative disease and cancer. importance to determine whether loss of CtBP function (A) Environmental stress and genetic mutations may induce underlies the neuronal cell death associated with dis- neuronal apoptosis through diminished CtBP activity, ultimately eases such as ALS, PD, and Alzheimer disease. In the resulting in neuroinflammation and neuronal cell death. Thus, resto- context of HD and other neurodegenerative diseases ration of CtBP activity via ADIOL, a compound designed to stabilize CtBP dimers, a Bcl-3 mimetic, or a JNK inhibitor may offer novel for which loss of CtBP function may play a pivotal role approaches to treat neurodegenerative diseases. (B) Genomic and in neuronal apoptosis in the CNS, it would be a worthy environmental stressors may also enhance CtBP activity, resulting approach to perform a small molecule screen to identify in increased cellular survival, EMT, and ultimately carcinogenesis. compounds that function to stabilize the dimerization of Utilizing CtBP inhibitors such as MTOB, cisplatin, or cyclo-SGWTV- CtBPs, thereby increasing their stability and activity. Fur- VRMY may lead to reduced CtBP activity and tumor suppression. thermore, given the involvement of Rac GTPase in modulating the expression of CtBP in primary neurons, small molecule activators of a Rac-specific GEF (e.g., Tiam1) loss of CtBP function may contribute to neuronal cell may also promote increased CtBP expression to enhance death and ultimately neurodegenerative disease. Thus, neuronal survival. enhancing CtBP function in both neurons and glial cells In addition, the potential involvement of CtBPs in of the CNS offers a novel and largely unexplored thera- suppressing inflammation through binding and repress- peutic approach for the treatment of neurodegenerative ing the transcription of genes that are implicated in diseases. activation of the inflammatory response suggests that targeting CtBP activation in microglia and astrocytes may also offer promising results for the treatment of neurode- CtBPs as targets for cancer treatment generative disease. In the clinic, the ERβ-specific ligand, indazole-Cl, has already been demonstrated to reduce Increasing evidence supports a role for CtBPs in the inflammation in autoimmune encephalomyelitis (98), conversion of healthy cells to a neoplastic phenotype. and therefore, ERβ-specific ligands represent potential This occurs through regulation of several different path- therapeutic compounds for the treatment of neurodegen- ways including the effect of CtBP on the expression of erative diseases in which inflammation plays a patho- epithelial specific genes, as well as repression of tumor genic role. In conclusion, although relatively few studies suppressor genes. Therefore, therapeutic inhibition of have examined the function of CtBPs in maintaining neu- CtBP action may be a viable option for cancer treatments ronal survival to date, recent studies have revealed that (Figure 7B). T.R. Stankiewicz et al.: CtBPs in development and disease 505

MTOB is known to be cytotoxic to cancer cells and is treatment of these devastating diseases. Future pre-clini- a strong substrate for CtBP, thereby inhibiting the endog- cal studies will likely highlight CtBPs and their interacting enous actions of CtBP. Examination of cell death induced partners as novel therapeutic targets for the treatment of by CtBP inhibition with MTOB indicated that this treat- cancer and/or neurodegenerative disease. ment promotes cell death in various cancer cell lines through displacement of CtBP from the pro-apoptotic Bik promoter (115). MTOB has also been shown to be effective in suppressing ovarian cancer cell-line survival in a CtBP- Highlights dependent manner (121). Importantly, these findings seem –– CtBPs are well-described transcriptional corepressors. to be specific to malignant cells, which is a crucial factor –– CtBPs repress many pro-apoptotic genes. in the physiological elimination of cancer cells (115). –– Loss of CtBPs enhances sensitivity to apoptosis and Cisplatin is an alkylating agent commonly used as anoikis in non-neuronal cells. a chemotherapeutic agent in the treatment of cancer. –– Downregulation of CtBPs is sufficient to provoke neu- Evaluation of human lung cancer cell lines demonstrated ronal apoptosis. that cisplatin decreases CtBP levels in a JNK-dependent –– A reduction in CtBP function may contribute to neuro- manner (51). This interaction has proven to be important degenerative disease. in the development of chemotherapeutic agents, such as –– Enhanced expression of CtBPs can lead to EMT and cisplatin, which may partially exert its effects through may underlie the metastatic nature of anoikis-insen- HIPK2 activation (142). In conclusion, the importance of sitive tumor cells. CtBP involvement in cancer is highlighted by the develop- –– Enhanced CtBP function may contribute to the devel- ment of therapeutic interventions that directly target CtBP opment of certain cancers. actions. Furthermore, as CtBP is specifically dysregulated in tumor cell populations, and not in healthy cell populations, therapeutic targeting of CtBP dysregulation represents a unique approach to developing cancer cell-specific treatments. As studies continue into the mechanistic roles List of abbreviations of CtBP in cancer development, new insight will be given ADIOL 5-androsten-3β,17β-diol into additional ways to modulate aberrant CtBP function ALS amyotrophic lateral sclerosis in tumor cells. AMPK AMP-activated protein kinase APC adenomatous polyposis coli ARF alternative reading frame BKLF8 basic Krüppel-like factor 8 BRCA breast cancer Expert opinion CGNs cerebellar granule neurons CDIs cyclin-dependent kinase inhibitors CtBPs play a critical role in development, cellular survival, CtBP C-terminal binding protein and tumorigenesis. Although early studies highlighted an dCtBP Drosophila CtBP CtIPs CtBP-interacting proteins essential function for CtBPs in transcriptional corepres- ECM extracellular matrix sion, more recent data have highlighted the involvement EMT epithelial-to-mesenchymal transition of CtBPs in additional cellular functions such as transcrip- Evi-1 ecotropic virus integration site-1 tional activation and Golgi maintenance. At the cellular FAP familial adenomatous polyposis and organismal level, CtBPs have been well described as GEF guanine nucleotide exchange factor pro-survival molecules that are capable of repressing apo- HIC1 hypermethylated in cancer HCC hepatocellular carcinoma ptosis, and more specifically, anoikis. Nonetheless, the HD Huntington disease involvement of CtBPs in promoting cancer and the poten- HDAC histone deacetylase tial loss of CtBP function in neurodegenerative disease Hdm2/Mdm2 oncoprotein human double minute 2/mouse are relatively unexplored concepts. Thus, future studies double minute 2 should aim to elucidate the precise involvement of CtBP HIPK homeodomain interacting protein kinase 2 HMT histone methyltransferase dysregulation in in vitro and in vivo models of disease. In Htt huntingtin addition, inhibition or activation of CtBPs in cancer and JNK c-Jun N-terminal kinase neurodegenerative disease models, respectively, should LPS lipopolysaccharide be examined as a potential therapeutic approach for LSD1 lysine-specific demethylase 1 506 T.R. Stankiewicz et al.: CtBPs in development and disease

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and CtBP modulate the correlations between SNAIL, ZEB1, E-cadherin and vitamin D receptor in human colon carcinomas. Bionotes Int J Cancer 2006; 119: 2098–104. Trisha R. Stankiewicz 130. Postigo AA, Depp JL, Taylor JJ, Kroll KL. Regulation of Smad Research Service, Veterans Affairs Medical signaling through a differential recruitment of coactivators and Center, Denver, CO 80220, USA; and corepressors by ZEB proteins. EMBO J 2003; 22: 2453–62. Department of Biological Sciences and 131. Guaita S, Puig I, Franci C, Garrido M, Dominguez D, Batlle E, Eleanor Roosevelt Institute, University of Sancho E, Dedhar S, De Herreros AG, Baulida J. Snail induc- Denver, Denver, CO 80208, USA tion of epithelial to mesenchymal transition in tumor cells is accompanied by MUC1 repression and ZEB1 expression. J Biol Chem 2002; 277: 39209–16. 132. Eger A, Aigner K, Sonderegger S, Dampier B, Oehler S, Schreiber M, Berx G, Cano A, Beug H, Foisner R. DeltaEF1 Trisha R. Stankiewicz obtained her undergraduate degree in Biology is a transcriptional repressor of E-cadherin and regulates from the University of Denver in Denver, CO. She remained at the epithelial plasticity in breast cancer cells. Oncogene 2005; 24: University of Denver to complete her PhD research under the guid- 2375–85. ance of Dr. Daniel Linseman. Here, her work focused on understand- 133. Peña C, Garcia JM, Silva J, Garcia V, Rodriguez R, Alonso I, ing the involvement of Rho GTPases and C terminal binding proteins Millan I, Salas C, de Herreros AG, Munoz A, Bonilla F. in regulating neuronal survival. Dr. Stankiewicz received her PhD in E-cadherin and vitamin D receptor regulation by SNAIL and Biological Sciences from the University of Denver in June, 2014 and ZEB1 in colon cancer: clinicopathological correlations. Hum is currently a postdoctoral fellow in the Department of Pathology at Mol Genet 2005; 14: 3361–70. Stanford University School of Medicine. 134. Spoelstra NS, Manning NG, Higashi Y, Darling D, Singh M, Shroyer KR, Broaddus RR, Horwitz KB, Richer JK. The transcrip- Josie J. Gray tion factor ZEB1 is aberrantly expressed in aggressive uterine Research Service, Veterans Affairs Medical cancers. Cancer Res 2006; 66: 3893–902. Center, Denver, CO 80220, USA; and 135. Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in Department of Biological Sciences and tumour progression: an alliance against the epithelial pheno- Eleanor Roosevelt Institute, University of type? Nat Rev Cancer 2007; 7: 415–28. Denver, Denver, CO 80208, USA 136. van Grunsven LA, Taelman V, Michiels C, Opdecamp K, Huylebroeck D, Bellefroid EJ. deltaEF1 and SIP1 are differentially expressed and have overlapping activities during Xeno- pus embryogenesis. Dev Dyn 2006; 235: 1491–500. Josie J. Gray obtained her undergraduate degree in Molecular, 137. Alpatov R, Shi Y, Munguba GC, Moghimi B, Joo JH, Bungert J, Cellular, and Developmental Biology (MCDB), Biochemistry, and Sugrue SP. Corepressor CtBP and nuclear speckle protein Pnn/ Psychology from the University of Colorado, Boulder. Upon gradu- DRS differentially modulate transcription and splicing of the ation, she worked for several years in various fields of pathology, E-cadherin gene. Mol Cell Biol 2008; 28: 1584–95. most notably cancer genetics and diagnostics, before returning to 138. Deng Y, Deng H, Bi F, Liu J, Bemis LT, Norris D, Wang XJ, graduate school at the University of Denver. There, she studied Zhang Q. MicroRNA-137 targets carboxyl-terminal binding mitochondrial involvement in neurodegeneration and specifically protein 1 in melanoma cell lines. Int J Biol Sci 2011; 7: 133–7. examined mitochondrial dynamics during apoptosis, and regulation 139. Sood R, Talwar-Trikha A, Chakrabarti SR, Nucifora G. MDS1/ of the transactivating response DNA-binding protein-43 (TDP-43) in EVI1 enhances TGF-beta1 signaling and strengthens its amyotrophic lateral sclerosis (ALS). Dr. Gray received her PhD in growth-inhibitory effect but the leukemia-associated fusion Biological Sciences from the University of Denver in 2014. protein AML1/MDS1/EVI1, product of the t(3;21), abrogates growth-inhibition in response to TGF-beta1. Leukemia 1999; 13: Aimee Winter 348–57. Department of Biological Sciences and 140. Wang R, Asangani IA, Chakravarthi BV, Ateeq B, Lonigro RJ, Eleanor Roosevelt Institute, University of Cao Q, Mani RS, Camacho DF, McGregor N, Schumann TE, Denver, Denver, CO 80208, USA Jing X, Menawat R, Tomlins SA, Zheng H, Otte AP, Mehra R, Siddiqui J, Dhanasekaran SM, Nyati MK, Pienta KJ, Palanisamy N, Kunju LP, Rubin MA, Chinnaiyan AM, Varambally S. Role of transcriptional corepressor CtBP1 in prostate cancer progression. Neoplasia 2012; 14: 905–14. 141. Deng H, Liu J, Deng Y, Han G, Shellman YG, Robinson SE, Tentler JJ, Robinson WA, Norris DA, Wang XJ, Zhang Q. CtBP1 Aimee Winter attended the University of Denver for her under- is expressed in melanoma and represses the transcription of graduate education where she earned a Bachelor’s of Science in p16INK4a and Brca1. J Invest Dermatol 2013; 133: 1294–301. Molecular Biology. Following graduation, she joined the Department 142. Di Stefano V, Soddu S, Sacchi A, D’Orazi G. HIPK2 contributes of Biological Sciences at the University of Denver as a gradu- to PCAF-mediated p53 acetylation and selective transactivation ate student to pursue her PhD in Cell and Molecular Biology. A of p21Waf1 after nonapoptotic DNA damage. Oncogene 2005; fourth year PhD candidate, she studies under Dr. Daniel Linseman 24: 5431–42. researching pre-clinical therapeutic candidates for the treatment of T.R. Stankiewicz et al.: CtBPs in development and disease 511 amyotrophic lateral sclerosis (ALS). Currently, her work focuses on elucidating the neuroprotective and anti-inflammatory mechanisms of nutraceutical compounds both in vitro and in vivo in order to characterize and identify new therapeutic agents for further pre- clinical evaluation.

Daniel A. Linseman Research Service, Veterans Affairs Medical Center, Research 151, 1055 Clermont Street, Denver, CO 80220, USA; Department of Biological Sciences and Eleanor Roosevelt Institute, University of Denver, Denver, CO 80208, USA; and Division of Clinical Pharmacology and Toxicology, Department of Medicine and Neuroscience Program, University of Colorado Denver, Aurora, CO 80045, USA, [email protected]

Daniel Linseman’s research is focused on elucidating molecular mechanisms of neuronal cell death in degenerative disorders with a particular emphasis on amyotrophic lateral sclerosis (Lou Geh- rig’s disease). Biochemical, immunofluorescence, and molecular biological techniques are used in conjunction with in vitro neuronal systems and transgenic mouse models to examine the roles of Rho family GTPase signaling, mitochondrial oxidative stress, and intrinsic apoptosis in neurodegeneration. Specific projects include inves- tigating the role of Rho family GTPase dysregulation in neuronal cell death, involvement of Bcl-2 family proteins in the regulation of mitochondrial susceptibility to oxidative stress, and studies on the antioxidant and neuroprotective properties of a variety of natural products (e.g., green tea catechins and berry anthocyanins). Dr. Lin- seman enjoys mentoring students and currently has 3 PhD students and 5 undergraduate honors students in his two laboratories at the Denver VA Medical Center and the University of Denver where he has joint appointments.